Mechanical Withdrawal Threshold

The up-down method was used in this study (Chaplan et al., 1994). Von Frey filaments (Stoelting Co., Thermo, Gilroy, CA, United States) with forces of 0.4, 0.6, 1, 2, 4, 6, 8, 15, and 26 g were used. The rats were placed in a clear plastic cage for 30 min before assessment every day for three continuous days to allow acclimation to the environment. A von Frey filament with a force of 4 g was first applied to the central surface of the hind paw (avoiding the footpad) until the filament bent into an “S” shape, and it was maintained there for 6 s. A filament of a greater or lesser force was then chosen depending on whether the response was negative or positive. The responses were recorded as X or O. The pain threshold was calculated according to the following formula: Mechanical withdrawal threshold (MWT) (g) = [10^{(Xf+ κδ)}]/10,000, where “Xf” is the force of the last hair test, the “κ” value is obtained from the k-value table, and “δ” is the average value of the difference between the logarithm of hairs of each force, which is approximately equal to 0.231.

The nociceptive behaviors were quantified during the 8 o’clock to 17 o’clock during the day. And the experimenters were blind to the experimental design.